EP0726941B1 - Procedes de therapie (ex-vivo) recourant a des cellules presentatrices d'antigenes porteurs de peptides pour activer des ctl - Google Patents

Procedes de therapie (ex-vivo) recourant a des cellules presentatrices d'antigenes porteurs de peptides pour activer des ctl Download PDF

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EP0726941B1
EP0726941B1 EP94924539A EP94924539A EP0726941B1 EP 0726941 B1 EP0726941 B1 EP 0726941B1 EP 94924539 A EP94924539 A EP 94924539A EP 94924539 A EP94924539 A EP 94924539A EP 0726941 B1 EP0726941 B1 EP 0726941B1
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cells
antigen presenting
peptide
presenting cells
peptides
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EP0726941A4 (fr
EP0726941A1 (fr
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Esteban Celis
Ralph Kubo
Horacio Serra
Van Tsai
Peggy Wentworth
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Epimmune Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464484—Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE
    • A61K39/464486—MAGE
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5158—Antigen-pulsed cells, e.g. T-cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/11—Coculture with; Conditioned medium produced by blood or immune system cells
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to compositions and methods for making compositions for preventing or treating a number of pathological states such as viral diseases and cancer through ex vivo therapy.
  • it provides methods for inducing cytotoxic T lymphocytes (CTL) using antigen presenting cells (APC) with a peptide of choice bound to selected major histocompatibility complex (MHC) molecules.
  • CTL cytotoxic T lymphocytes
  • APC antigen presenting cells
  • MHC major histocompatibility complex
  • Cytotoxic T cells or CD8 cells as they are also known, represent the main line of defense against viral infections. CTLs specifically recognize and kill cells which are infected by a virus. The T cell receptors on the surface of CTLs cannot recognize foreign antigens directly. In contrast to antibodies, antigen must first be presented to the T cell receptors for activation to occur.
  • MHC major histocompatibility complex
  • HLA human leukocyte antigen
  • MHC molecules are classified as either Class I, Class II or class III molecules.
  • Class II MHC molecules are expressed primarily on cells involved in initiating and sustaining immune responses, such as T lymphocytes, B lymphocytes, macrophages, etc.
  • Class II MHC molecules are recognized by helper T lymphocytes and induce proliferation of helper T lymphocytes and amplification of the immune response to the particular immunogenic peptide that is displayed.
  • Class I MHC molecules are expressed on almost all nucleated cells and are recognized by CTLs.
  • T cells that serve mainly as helper cells express CD4 and are primarily restricted to Class II molecules, whereas CD8-expressing cells, represented by cytotoxic effector cells, interact with Class I molecules.
  • the CTL recognizes the antigen in the form of a peptide fragment bound to the MHC class I molecules rather than the intact foreign antigen itself.
  • the antigen must normally be endogenously synthesized by the cell, and a portion of the protein antigen is degraded into small peptide fragments in the cytoplasm. Some of these small peptides translocate into a pre-Golgi compartment and interact with class I heavy chains to facilitate proper folding and association with the subunit ⁇ 2 microglobulin.
  • the peptide-MHC class I complex is then routed to the cell surface for expression and potential recognition by specific CTLs.
  • LAK cell therapy or TIL [tumor infiltrating lymphocytes] therapy
  • TIL tumor infiltrating lymphocytes
  • Adoptive T cell therapy of tumors Mechanisms operative in the recognition and elimination of tumor cells. Advances in Immunology 49:281. Melief, C. 1992. Tumor eradication by adoptive transfer of cytotoxic T lymphocytes. Adv. Cancer Research 58:14. 34. Riddell, S., K. Watanabe, J. Goodrich, C. Li, M. Agha, P. Greenberg. 1992. Restoration of viral immunity in immunodeficient humans by the adoptive transfer of T cell clones. Science 257:238. ).
  • This invention is directed to methods of activating cytotoxic T cells (CD8 cells) in vitro.
  • the methods of activating CD8 cells comprise: dissociating bound peptides from class I MHC molecules on antigen presenting cells using a mild acid treatment; associating selected immunogenic peptides with the class I MHC molecule on the antigen presenting cell; and incubating the antigen presenting cells with the cytotoxic T cells in the presence of a growth factor, thereby producing activated cytotoxic T cells.
  • the methods of the present invention are capable of generating empty MHC class I molecules on antigen presenting cells and in turn inducing CTL and affecting killing of class I matched cells.
  • the antigen presenting cells having empty MHC class I molecules on their surface are capable of inducing cytotoxic T cells which are useful in the treatment of chronic infectious diseases and cancer.
  • this invention provides methods of producing empty MHC class I molecules on antigen presenting cells, loading those empty MHC class I molecules with selected immunogenic peptides, activating cytotoxic T cells which are specific for killing specific antigen targets.
  • This invention has broad therapeutic application in the treatment of cancers, certain immune diseases and viral diseases.
  • the method may further comprise: separating activated CTLs from the antigen presenting cells having the empty MHC class I molecule on its surface and suspending the activated CTLs in an acceptable carrier or excipient as a pharmaceutical composition; for administering to a patient having the disease.
  • peptide is used interchangeably with “oligopeptide” in the present specification to designate a series of residues, typically L-amino acids, connected one to the other typically by peptide bonds between the alpha-amino and carbonyl groups of adjacent amino acids.
  • immunogenic peptide is a peptide which comprises an allele-specific motif such that the peptide will bind the MHC allele and be capable of inducing a CTL response.
  • immunogenic peptides are capable of binding to an appropriate class I MHC molecule and inducing a cytotoxic T cell response against the antigen from which the immunogenic peptide is derived.
  • residue refers to an amino acid or amino acid mimetic incorporated in an oligopeptide by an amide bond or amide bond mimetic.
  • the present invention relates to methods of enhancing the immune response to various diseases using ex vivo therapy.
  • the general approach of the invention comprises isolation of peripheral blood mononuclear cells (PBMCs) from a patient, loading a desired immunogenic peptide into the binding pockets of MHC class I molecules on the surface of antigen presenting cells (APCs), incubating the APCs with precursor CTLs in the sample to induce proliferation of CTLs recognizing the peptide, and using the CTLs to identify antigenic epitopes and by expanding their numbers introduce the activated CTLs into the patient.
  • PBMCs peripheral blood mononuclear cells
  • APCs antigen presenting cells
  • the procedures of the present invention depend in part upon the determination of epitopes recognized by CTLs capable of eliminating target infected cells.
  • One approach to identification of these epitopes is the identification of allele-specific peptide motifs associated with a particular disease for human Class I MHC allele subtypes.
  • the MHC class I antigens are encoded by the HLA-A, B, and C loci.
  • HLA-A and B antigens are expressed at the cell surface at approximately equal densities, whereas the expression of HLA-C is significantly lower (perhaps as much as 10-fold lower).
  • Each of these loci have a number of alleles.
  • a large number of cells with defined MHC molecules, particularly MHC Class I molecules, are known and readily available. These cells can be used to identify particular allele specific motifs associated with target diseases.
  • the allele-specific motifs are then used to define T cell epitopes from any desired antigen, particularly those associated with human viral diseases or cancers, for which the amino acid sequence of the potential antigen targets is known. This general approach is described in detail in copending and commonly assigned applications U.S.S.N. 07/926,666 and U.S.S.N. 08/027,146.
  • PSA prostate specific antigen
  • HBVc hepatitis B core
  • HBVp hepatitis C antigens
  • Epstein-Barr virus antigens Epstein-Barr virus antigens
  • melanoma antigens e.g., MAGE-1
  • HAV human immunodeficiency virus
  • HPV human papilloma virus
  • HSV herpes simplex virus
  • c-Erb B 2 , CEA, p 53-breast/ovary c-Erb B 2 , CEA, p 53-breast/ovary
  • motifs specific for different class I alleles allows the identification of potential peptide epitopes from an antigenic protein whose amino acid sequence is known. Typically, identification of potential peptide epitopes is initially carried out using a computer to scan the amino acid sequence of a desired antigen for the presence of motifs. The epitopic sequences are then synthesized. The capacity to bind MHC Class molecules is measured in a variety of different ways using, for example, purified class I molecules and radioiodinated peptides and/or cells expressing empty class I molecules by, for instance, immunofluorescent staining and flow microfluorimetry, peptide-dependent class I assembly assays, and inhibition of CTL recognition by peptide competition.
  • peptides that test positive in the MHC class I binding assay are assayed for the ability of the peptides to induce specific primary or secondary CTL responses in vitro.
  • antigen-presenting cells that have been incubated with a peptide can be assayed for the ability to induce CTL responses in responder cell populations.
  • antigen-presenting cells can be normal cells such as peripheral blood mononuclear cells or dendritic cells (Inaba, et al., J. Exp. Med., 166:182 (1987); Boog, Eur. J. Immunol., 18:219 [1988]).
  • mutant mammalian cell lines that are deficient in their ability to load class I molecules with internally processed peptides, such as the mouse cell lines RMA-S (Kärre, et al.. Nature, 319:675 (1986); Ljunggren, et al., Eur. J. Immunol., 21:2963-2970 (1991)), and the human somatic T cell hybridoma, T-2 (Cerundolo, et al., Nature, 345:449-452 (1990)) and which have been transfected with the appropriate human class I genes are conveniently used, when peptide is added to them, to test for the capacity of the peptide to induce in vitro primary CTL responses.
  • RMA-S mouse cell lines
  • T-2 human somatic T cell hybridoma
  • MHC-peptide complexes are preferable for inducing a primary response since the density of MHC-peptide complexes on the surface of the antigen presenting cell will be greater.
  • Other eukaryotic cell lines which could be used include various insect cell lines such as mosquito larvae (ATCC cell lines CCL 125, 126, 1660, 1591, 6585, 6586), silkworm (ATTC CRL 8851), armyworm (ATCC CRL 1711), moth (ATCC CCL 80) and Drosophila cell lines such as a Schneider cell line that have been transfected with the appropriate human class I MHC allele encoding genes and the human B 2 microglobulin genes.
  • immunogenic peptides comprising the motif required for MHC binding and the epitope recognized by the CTL are synthesized.
  • the immunogenic peptides can be prepared synthetically, or by recombinant DNA technology or isolated from natural sources such as whole viruses or tumors.
  • One of skill will recognize that the immunogenic peptides can be a variety of lengths, either in their neutral (uncharged) forms or in forms which are salts, and either free of modifications such as glycosylation, side chain oxidation, or phosphorylation or containing these modifications, subject to the condition that the modification not destroy the biological activity of the polypeptides as herein described.
  • the peptide will be as small as possible while still maintaining substantially all of the biological activity of the large peptide.
  • Peptides having the desired activity may be modified as necessary to provide certain desired attributes, e.g., improved pharmacological characteristics, while increasing or at least retaining substantially all of the biological activity of the unmodified peptide to bind the desired MHC molecule and activate the appropriate T cell.
  • the peptides may be subject to various changes, such as substitutions, either conservative or non-conservative, where such changes might provide for certain advantages in their use, such as improved MHC binding.
  • conservative substitutions is meant replacing an amino acid residue with another which is biologically and/or chemically similar, e.g., one hydrophobic residue for another, or one polar residue for another.
  • substitutions include combinations such as Gly, Ala; Val, Ile, Leu, Met; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe, Tyr.
  • the effect of single amino acid substitutions may also be probed using D-amino acids. Such modifications may be made using well known peptide synthesis procedures.
  • the peptides of the invention can be prepared in a wide variety of ways. Because of their relatively short size, the peptides can be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Stewart and Young, Solid Phase Peptide Synthesis, 2d. ed., Pierce Chemical Co. (1984), supra.
  • recombinant DNA technology may be employed wherein a nucleotide sequence which encodes an immunogenic peptide of interest is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression.
  • a nucleotide sequence which encodes an immunogenic peptide of interest is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression.
  • the immunogenic peptides are then used to activate CTL ex vivo.
  • the ex vivo therapy methods of the present invention and pharmaceutical compositions thereof are useful for treatment of mammals, particularly humans, to treat and/or prevent viral infection, immune disorders and cancer.
  • diseases which can be treated using the ex vivo therapy methods of the invention include prostate cancer, hepatitis B, hepatitis C, AIDS, renal carcinoma, cervical carcinoma, lymphoma, CMV, condyloma acuminatum breast and ovarian cancer, colon, lung cancer and HSV.
  • therapy should begin at the first sign of viral infection or the detection or surgical removal of tumors or shortly after diagnosis in the case of acute infection. This is followed by boosting levels of CTL at least until symptoms are substantially abated and for a period thereafter. In chronic infection, loading doses followed by boosting doses may be required.
  • Treatment of an infected individual with the products of the methods of the invention may hasten resolution of the infection in acutely infected individuals. For those individuals susceptible (or predisposed) to developing chronic infection the methods are useful for preventing the evolution from acute to chronic infection. Where the susceptible individuals are identified prior to or during infection, the compositions can be targeted to them, minimizing the need for administration to a larger population.
  • the products of the methods of the present invention can also be used for the treatment of chronic infection and to stimulate the immune system to eliminate virus-infected cells in carriers.
  • CTL responses to a particular pathogen are induced by incubating in tissue culture the patient's CTL precursor cells (CTLp) together with a source of antigen-presenting cells (APC) loaded with the appropriate immunogenic peptide.
  • CTLp CTL precursor cells
  • APC antigen-presenting cells
  • the cells are infused back into the patient, where they will destroy their specific target cell (an infected cell or a tumor cell).
  • Infusion of the cells into the patient may include a T cell growth factor such as interleukin 2 (IL-2).
  • IL-2 interleukin 2
  • the culture of stimulator cells is maintained in an appropriate serum-free medium which may include one or more growth factors such as IL-2, IL-4, IL-7 and IL-12.
  • Peripheral blood lymphocytes are conveniently isolated following simple venipuncture or leukapheresis of normal donors or patients and used as the responder cell sources of CTLp.
  • the appropriate APC are incubated with 10-100 ⁇ M of peptide in serum-free media for 4 hours under appropriate culture conditions.
  • the peptide-loaded APC are then incubated with the responder cell populations in vitro for 7 to 10 days under optimized culture conditions.
  • APC expressing empty MHC would be used to stimulate naive CTLp. In this case the CTL would be stimulated more frequently (1-2 times).
  • Positive CTL activation can be determined by assaying the cultures for the presence of CTLs that kill radiolabeled target cells, both specific peptide-pulsed targets as well as target cells expressing endogenously processed form of the relevant virus or tumor antigen from which the peptide sequence was derived.
  • Specificity and MHC restriction of the CTL of a patient can be determined by a number of methods known in the art. For instance, CTL restriction can be determined by testing against different peptide loaded target cells expressing human MHC class I alleles shared with the HLA phonotype of the donor CTL. The peptides that test positive in the MHC binding assays and give rise to specific CTL responses are identified as immunogenic peptides.
  • the induction of CTL in vitro requires the specific recognition of peptides that are bound to allele specific MHC class I molecules on APC.
  • the number of specific MHC/peptide complexes per APC determines the level of stimulation of CTL, particularly during the primary immune response. While small amounts of peptide/MHC complexes per cell are sufficient to render a cell susceptible to lysis by CTL, or to stimulate a secondary CTL response, the successful activation of a CTLp during primary response requires a significantly higher number of MHC/peptide complexes.
  • mutant cell lines capable of expressing empty MHC do not exist for every human MHC allele, it is advantageous to use a technique to remove endogenous MHC-associated peptides from the surface of APC, followed by loading the resulting empty MHC molecules with the immunogenic peptides of interest.
  • the use of non-transformed (non-tumorigenic), non-infected cells, and preferably, autologous cells of patients as APC is desirable for the design of CTL induction protocols directed towards development of ex vivo CTL therapies.
  • This present invention provides novel methods generating empty class I MHC which can then be loaded with an appropriate immunogenic peptide by stripping the endogenous MHC-associated peptides from the surface of APC or through cold temperature incubation (37°C ⁇ 26°C)followed by the loading of desired peptides.
  • a stable MHC class I molecule is a trimeric complex formed of the following elements: 1) a peptide usually of 8 - 10 residues, 2) a transmembrane heavy polymorphic protein chain which bears the peptide-binding site in its ⁇ 1 and ⁇ 2 domains, and 3) a non-covalently associated non-polymorphic light chain, ⁇ 2 microglobulin. Removing the bound peptides and/or dissociating the ⁇ 2 microglobulin from the complex renders the MHC class I molecules nonfunctional and unstable, resulting in rapid degradation at 37°C. Almost all MHC class I molecules isolated from PBMCs have endogenous peptides bound to them. Therefore, the first step to prepare APC for primary CTL induction is to remove all endogenous peptides bound to MHC class I molecules on the APC without causing degradation or cell death before exogenous peptides can be added.
  • Two possible ways to generate free MHC class I molecules include lowering the culture temperature from 37°C to 26°C overnight to allow MHC class I without peptides to be expressed and stripping the endogenous peptides from the cell using a mild acid treatment.
  • the mild acid treatment releases previously bound peptides into the extracellular environment allowing new exogenous peptides to bind to the empty class I molecules.
  • the overnight cold-temperature incubation at 26°C which may slow the cell's metabolic rate enables expression of stable empty class I molecules which then bind exogenous peptides efficiently. It is also likely that cells not actively synthesizing MHC molecules (e.g., resting PBMC) would not produce high amounts of empty surface MHC molecules by the cold temperature procedure.
  • Extraction of the peptides is accomplished by harsh acid stripping using trifluoroacetic acid, pH 2, or acid denaturation of the immunoaffinity purified class I-peptide complexes. These methods are not feasible for CTL induction, since it is important to remove the endogenous peptides while preserving APC viability and an optimal metabolic state which is critical for antigen presentation.
  • Mild acid solutions of pH 3 such as glycine or citrate-phosphate buffers have been used to identify endogenous peptides and to identify tumor associated T cell epitopes (31. Storkus, W., H. Zeh, R. Salter, and M. Lotze. 1993.
  • T cell epitopes Rapid isolation of class I-presented peptides from viable cells by mild acid elution [submitted]).
  • the treatment is especially effective, in that only the MHC class I molecules are destabilized (and associated peptides released), while other surface antigens remain intact, including MHC class II molecules.
  • MHC class II molecules 16. Suguwara, S., T. Abo, and K. Kumagai. 1987. A simple method to eliminate the antigenicity of surface class I MHC molecules from the membrane of viable cells by acid treatment at pH 3. J. Immunol. Meth. 100:83).
  • treatment of cells with the mild acid solutions do not affect the cell's viability or metabolic state.
  • the mild acid treatment is rapid since the stripping of the endogenous peptides occurs in two minutes at 4°C and the APC is functional after the appropriate peptides are loaded.
  • the technique is utilized herein to make peptide-specific APCs for the generation of primary antigen-specific CTL.
  • the resulting APCs are efficient in inducing peptide-specific CTL.
  • an amount of antigenic peptide is added to the APCs or stimulator cell culture, of sufficient quantity to become loaded onto the human Class I molecules to be expressed on the surface of the APCs.
  • a sufficient amount of peptide is an amount that will allow about 200 or more human Class I MHC molecules loaded with peptide to be expressed on the surface of each stimulator cell.
  • the stimulator cells are incubated with 5-100 ⁇ g/ml peptide.
  • the CTLs are then incubated in culture with the appropriate APCs for a time period sufficient to activate the CTLs.
  • the CTLs are activated in an antigen-specific manner.
  • the ratio of precursor CTLs to APCs may vary from individual to individual and may further depend upon variables such as the amenability of an individual's lymphocytes to culturing conditions and the nature and severity of the disease condition or other condition for which the within-described treatment modality is used.
  • the CTL:APC (i.e. responder to stimulator) ratio is in the range of about 10:1 to 100:1.
  • the CTL/APC culture may be maintained for as long a time as is necessary to stimulate a therapeutically useable or effective number of CTL.
  • Activated CTL may be effectively separated from the APC using one of a variety of known methods.
  • monoclonal antibodies specific for the APCs, for the peptides loaded onto the stimulator cells, or for the CTL (or a segment thereof) may be utilized to bind their appropriate complementary ligand.
  • Antibody-tagged cells may then be extracted from the admixture via appropriate means, e.g., via well-known immunoprecipitation or immunoassay methods.
  • Effective, cytotoxic amounts of the activated CTLs can vary between in vitro and in vivo uses, as well as with the amount and type of cells that are the ultimate target of these killer cells. The amount will also vary depending on the condition of the patient and should be determined via consideration of all appropriate factors by the practitioner. Preferably, however, about 1 X 10 6 to about 1 X 10 12 , more preferably about 1 X 10 8 to about 1 X 10 11 , and even more preferably, about 1 X 10 9 to about 1 X 10 10 activated CTLS are utilized for adult humans, compared to about 5 X 10 6 - 5 X 10 7 cells used in mice.
  • the activated CTLS may be harvested from the cell culture prior to administration of the cells to the individual being treated. It is important to note, however, that unlike other present treatment modalities, the present method uses a cell culture system that does not contain transformed or tumor cells. Therefore, if complete separation of antigen-presenting cells and activated CTLS is not achieved, there is no inherent danger known to be associated with the administration of a small number of stimulator cells, whereas administration of mammalian tumor-promoting cells may be extremely hazardous.
  • the APC uses the APC generated by the in vitro techniques of this application for therapy against CTL in vivo.
  • the APC are a patient's cells (e.g., the peripheral blood cells) which are stripped of their natural antigenic peptides and loaded with a peptide of choice which is conjugated to a toxin (e.g. ricin A chain or pseudomonas toxin).
  • the APCs are then re-introduced into the patient, where they will be bound by the endogenous CTLs that are specific for the antigenic peptide.
  • the coupled toxin will kill the activated CTL that are harmful i.e. those which stimulate transplant rejection after it binds the APC.
  • Such directed CTL killing is broadly useful for treating tissue-transplantation rejection and auto-immune disorders, which are mediated through CTL.
  • the treatment regime will vary depending upon the specific disorder to be treated and the judgement of the treating physician.
  • CTL Cytotoxic T Lymphocytes
  • PBMC Peripheral blood mononuclear cells
  • the isolated and purified PBMC are co-cultured with an appropriate number of APC expressing empty MHC molecules, previously incubated ("pulsed") with an appropriate amount of synthetic peptide (containing the HLA binding motif and the sequence of the antigen in question).
  • PBMC are usually incubated at 1-3 X 10 6 cells/ml in culture medium such as RPMI-1640 (with autologous serum or plasma) or the serum-free medium AIM-V (Gibco).
  • APC are usually used at concentrations ranging from 1X10 4 to 1X10 6 cells/ml, depending on the type of cell used.
  • Possible sources of APC include: autologous PBMCs, SAC-I activated PBMCs, PHA blasts; autologous dendritic cells (DC) which are isolated from PBMC and purified as described (Inaba, et al., J. Exp. Med., 166:182 (1987)); and mutant and genetically engineered mammalian cells such as the mouse RMA-S cell line or the human T2 cell line transfected with the appropriate MHC genes that express "empty" HLA molecules which are syngeneic to the patient's allelic HLA form).
  • DC dendritic cells
  • APC containing empty HLA molecules are known to be potent inducers of CTL responses, possibly because the peptide can associate more readily with empty MHC molecules than with MHC molecules which are occupied by other peptides (DeBruijn, et al., Eur. J. Immunol ., 21:2963-2970 (1991)).
  • the APC are gamma irradiated with an appropriate dose (using, e.g., radioactive cesium or cobalt) to prevent their proliferation and to facilitate the expansion of the CTLp.
  • an appropriate dose using, e.g., radioactive cesium or cobalt
  • the mixture cultures, containing PBMC, APC and peptide are kept in an appropriate culture vessel such as plastic T-flasks, gas-permeable plastic bags, or roller bottles, at 37° centigrade in a humid air/CO 2 incubator.
  • an appropriate culture vessel such as plastic T-flasks, gas-permeable plastic bags, or roller bottles, at 37° centigrade in a humid air/CO 2 incubator.
  • the resulting effector CTL can be further expanded, by the addition of recombinant growth factors such as interleukin-2 (IL-2), interleukin-4 (IL-4), or interleukin-7 (IL-7) to the cultures.
  • An expansion culture can be kept for an additional 5 to 12 days, depending on the numbers of effector CTL required for a particular patient.
  • expansion cultures may be performed using hollow fiber artificial capillary systems (Cellco), where larger numbers of cells (up to 1X10 11 ) can be maintained.
  • Cellco hollow fiber artificial capillary systems
  • the cytotoxic activity of the resulting CTL can be determined by a standard 51 Cr-release assay (Biddison, W.E. 1991, Current Protocols in Immunology, p7,17.1-7.17.5, Ed. J. Coligan et al., J. Wiley and Sons, New York), using target cells that express the appropriate HLA molecule, in the presence and absence of the immunogenic peptide. Viability is determined by the exclusion of trypan blue dye by live cells. Cells are tested for the presence of endotoxin by conventional techniques.
  • the presence of bacterial or fungal contamination is determined by appropriate microbiological methods (chocolate agar, etc.). Once the cells pass all quality control and safety tests, they are washed and placed in the appropriate infusion solution (Ringer/glucose lactate/human serum albumin) which may include a T-cell growth factor such as IL-2 and infused intravenously into the patient.
  • appropriate infusion solution Rost agar, etc.
  • T-cell growth factor such as IL-2
  • This example demonstrates the use of cold temperature incubation and acid stripping for generation of empty MHC class I molecules to enable peptide loading method to prepare effective HLA-allele-specific antigen presenting cells (APC) for use in diagnostic or ex vivo therapy applications.
  • the APC in this example were used to sensitize precursor cytotoxic T lymphocytes for the development of antigen-specific cytotoxic cells. This was accomplished using either staphylococcus aureus cowan I SAC I activated PBMC, phytohemagglutinin (PHA) T-cell blasts or peripheral blood mononuclear cells (PBMC) as APC in the HLA-A2.1 and HLA-A1 systems. The results are applicable to other APC and to the other MHC alleles. Culture Medium.
  • PHA blasts and CTL inductions were done in RPMI 1640 + Hepes + glutamine (Gibco) supplemented with 2 mM L-glutamine (Irvine Scientific), 50 ⁇ g/ml gentamicin (Gibco), and 5% heat inactivated pooled human Type AB serum (Gemini Bioproducts) [RPMI/5% HS].
  • EBV transformed lymphoblastoid cell lines were maintained in RPMI 1640 + Hepes + glutamine (BioWhittaker) supplemented with L-glutamine and gentamicin as above and 10% heat inactivated fetal calf serum (Irvine Scientific) [RPMI/10% FCS].
  • Chromium release assays were performed in RPMI/10% FCS. Cytokines. Recombinant human interleukin-2 (rIL-2) (Sandoz) was used at a final concentration of 10 U/ml. Recombinant human interleukin-7 (rIL-7) (Genzyme) was used at a final concentration of 10 ng/ml.
  • Cultured Cell Lines JY, a HLA A2.1 expressing human EBV-transformed B-cell line, was grown in RPMI/10% FCS. K562, a NK cell sensitive erythroblastoma line was grown in RPMI/10% FCS. K562 was used to reduce background killing by NK and LAK cells in the chromium release assays. Peptides.
  • the immunogenic peptides used in these studies were synthesized as described above using motifs for HLA alleles for specific target antigens as described in detail in copending and commonly assigned applications U.S.S.N. 07/926,666 and U.S.S.N. 08/027,146 and their sequences are shown in Table 1.
  • Peptides were routinely dissolved in 100% DMSO at 20 mg/ml, aliquoted, and stored at -20°C. Isolation of Peripheral Blood Mononuclear Cells (PBMC).
  • PBMC Peripheral Blood Mononuclear Cells
  • the interface containing the PBMCs was collected using a transfer pipet (two interfaces per 50cc tube) and washed three times with 50 ml serum free RPMI (1700, 1500, and 1300 rpm for 10 minutes. Freezing and Thawing PBMC.
  • PBMC were frozen at 30 x 10 6 cells/ml of 90% FCS + 10% DMSO (Sigma) in 1 ml aliquots using cryovials (Nalge). Cryovials were placed in Cryo 1°C freezing containers (Nalge) containing isopropanol (Fisher) and placed at -70°C from 4 hours (minimum) to overnight (maximum). Isopropanol was changed after every 5 uses.
  • CD4+ lymphocyte depletion was performed using antibody-coated, flasks: MicroCELLector T-150 flasks for the selection of CD4+ cells (Applied Immune Sciences) were washed according to the manufacturer's instructions with 25 ml PBS CMF (calcium magnesium free) + 1 mM EDTA (Sigma) by swirling flasks for 30 sec followed by incubation for 1 hour at room temperature on a flat surface. Buffer was aspirated and flasks were washed 2 additional times by shaking the flasks for 30 seconds and maintaining coverage of the binding surface. To each washed flask, 25 ml culture medium were added and incubated for 20 minutes at room temperature on a flat surface.
  • PBMC peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • Culture medium was aspirated from the flask and then the cell suspension was gently added to the MicroCELLector.
  • Flasks containing the cells were incubated for 1 hour at room temperature on a flat surface. At the end of the incubation, the flask was gently rocked from side to side for 10 seconds to resuspend the nonadherent cells.
  • Nonadherent CD4+ T cell depleted cells were harvested and then flasks were washed twice with PBS CMF to collect the nonadherent cells.
  • Harvested CD4+ T cell depleted cells were pelleted by centrifugation and resuspended in culture medium.
  • Generation of PHA Blasts PBMC were isolated using the standard Ficoll-Paque protocol. Frozen cells were washed twice before use. Cells were cultured at 2 x 10 6 /ml in RPMI/5% HS containing 1 ⁇ g/ml PHA (Wellcome) and 10 U/ml rIL-2. PHA blasts were maintained in culture medium containing 10 U/ml rIL-2 with feeding and splitting as needed.
  • PHA blasts were used as APCs on day 6 of culture. Generation of empty class I molecules and peptide loading was only performed by the acid strip method when using PBMCs as APCs. Acid Stripping/Peptide Loading of PBMC and PHA Blasts. PBMC were isolated using the Ficoll-Paque protocol. When using frozen cells, PBMC were washed twice before using. PHA blasts were prepared as previously described and washed twice before using. Once cells were prepared, they were washed once in cold sterile 0.9% NaCl (J.T. Baker) + 1% BSA.
  • the cells were resuspended at 10 7 /ml in cold sterile citrate-phosphate buffer [0.13 M citric acid (J.T. Baker), 0.06 M sodium phosphate monobasic (Sigma) pH 3, 1% BSA, 3 ⁇ g/ml ⁇ 2 microglobulin (Scripps Labs)] and incubated for 2 minutes on ice.
  • cold sterile neutralizing buffer #1 [0.15 M sodium phosphate monobasic pH 7.5, 1% BSA, 3 ⁇ g/ml ⁇ 2 microglobulin, 10 ⁇ g/ml peptide] were added, and the cells were pelleted at 1500 rpm, 5 min at 4°C.
  • Cells were resuspended in 1 volume cold sterile neutralizing buffer #2 [PBS CMF, 1% BSA, 30 ⁇ g/ml DNAse, 3 ⁇ g/ml ⁇ 2 microglobulin, 40 ⁇ g/ml peptide] and incubated for 4 hours at 20°C. Cells were diluted with culture medium to approximately 5 x 10 6 /ml and irradiated with 6000 rads. Cells were then centrifuged at 1500 rpm for 5 minutes at room temperature and resuspended in culture medium. The acid stripped/peptide loaded cells were used immediately in the CTL induction cultures (below). Binding Assays Using Intact Cells and Radiolabelled Peptide.
  • JY cells were either acid stripped (i.e. treated with citrate-phosphate buffer and neutralizing buffer #1 as described above) or incubated at a reduced temperature. JY control cells were left untreated in tissue culture media. After treatment both cell populations were washed twice with serum free RPMI and loaded with 125 I-radiolabelled 941.01 (HBc 18-27) peptide (standard chloramine T iodination). To determine binding specificity, 2 x 10 6 cells were resuspended in 200 ⁇ l neutralizing buffer #2 (described above) containing 125 1-941.01 (10 5 cpms) +/- 100 ⁇ g unlabelled 941.01.
  • JY cells an HLA-A2.1 EBV-transformed B cell line
  • JY cells were preincubated at 26°C overnight or acid-stripped to remove the endogenous MHC-associated peptides and the loading of exogenous peptide was determined using a 125 I-radiolabelled HLA-A2.1 binding peptide.
  • the specificity of this reaction was determined by measuring the inhibition of labelled peptide binding using a cold peptide of the same sequence. Results presented in Table 2 demonstrate that acid-treatment of the cells increased significantly (approximately 10-fold) the amount of labelled peptide binding to the JY cells.
  • BB7.2 anti-HLA-A2
  • BB7.2 anti-HLA alpha chain-specific (9.12.1) monoclonal antibodies.
  • Controls for this experiment included the same cell population which was not treated at pH 3 (but treated with PBS buffer at pH 7.2), and cells treated with citrate-phosphate buffer (to strip the MHC) but neutralized in the absence of ⁇ 2 microglobulin and peptide.
  • Responder cells were resuspended in culture medium at 3 x 10 6 /ml and 1 ml of the responder cell suspension was dispensed into each well of a 24-well tissue culture plate (Falcon, Becton Dickinson). The plates were placed in the incubator at 37°C, 5% CO 2 until the stimulator population was ready. Once irradiated, stimulator APCs were resuspended in culture medium containing 20 ng/ml rIL-7 at 10 6 /ml for the PBMC, or at 3 x 10 5 /ml for the PHA blasts, 1 ml of stimulator cell suspension was added per well to the plates containing the responders.
  • Cells were pelleted and resuspended in culture medium at 4 x 10 6 /ml and 1 ml of cell suspension was added to each well of a 24-well tissue culture plate, and incubated for 2 hours at 37°C, 5% CO 2 . Nonadherent cells were removed by washing each well three times with serum free RPMI. After this step, a 0.5 ml culture medium containing 3 ⁇ g/ml ⁇ 2 microglobulin and 20 ⁇ g/ml total peptide was added to each well. APC were incubated for 2 hrs at 37°C, under 5% CO 2 with the peptide and ⁇ 2 microglobulin.
  • CTL were stimulated by SAC-I activated PBMCs as APC.
  • Cold temperature enhanced expression of empty MHC enabling loading of antigenic peptide to generate SAC-I activated PBMC APC.
  • This method presents an alternative protocol to the methods described above for the generation of the APC which are used to stimulate CTL.
  • This example also presents an alternative protocol for the stimulation of CTL by the APC.
  • Complete Culture Medium
  • the tissue culture medium used in this study consisted of RPMI 1640 with Hepes and L-glutamine (Gibco) (Biowhittaker) supplemented with 2 mM L-glutamine (Irvine Scientific), 0.5mM sodium pyruvate (Gibco), 100 U/100 ug/ml penicillin/streptomycin (Irvine), and 5% heat-inactivated Human Serum Type AB (RPMI/5% HS; Gemini Bioproducts).
  • Culture media used in the growth of EBV-transformed lines contained 10% heat-inactivated fetal calf serum (RPMI/10% FCS, Irvine) instead of human serum. Cytokines.
  • rIL-2 Recombinant human Interleukin-2 (rIL-2) and Interleukin-4 (rIL-4) were obtained from Sandoz and used at a final concentration of 10 U/ml and 10 ng/ml, respectively.
  • Peptides Peptides. Peptides were synthesized as described above and are described in Table 1. Peptides were routinely dissolved in 100% DMSO at 20 mg/ml, aliquoted, and stored at -70°C until used. Cell Lines.
  • JY, Steinlein, EHM, BVR, and KT3 are homozygous human EBV-transformed B cell lines expressing HLA A 2.1 , A 1 , A 3 , A 11 , and A 24 , respectively. They are grown in RPMI/10% FCS and used as targets in the CTL assays. K562, an NK cell sensitive, erythroblastoma line grown in RPMI/10% FCS, was used for reduction of background killing in the CTL assays.
  • Melanoma HLA A1+ cell lines either expressing the MAGE. antigen, mel 397 and mel 938 or those not expressing the MAGE antigen, mel 888, were also grown in RPMI/10% FCS.
  • PBMCs Peripheral Blood Mononuclear Cells
  • the interface between the Ficoll and the plasma containing the PBMCs was recovered with a transfer pipet (two interfaces per 50 ml tube) and washed three times with 50 ml of RPMI (1700, 1500, and 1300 RPM for 10 minutes). Cells were resuspended in 10-20 ml of culture medium, counted, and adjusted to the appropriate concentration. Freezing PBMCs. 30 million cells/tube (90% FCS/10% DMSO; Sigma) were inserted into a Nalgene Cryo 1°C Freezing Container containing isopropanol (Fisher) and placed at -70°C from 4 hrs (minimum) to overnight (maximum). The isopropanol was changed every five times.
  • Tubes were transferred to liquid nitrogen for long term storage. To thaw, PBMCs were continuously shaken in a 37°C water bath until the last crystal was almost thawed (tubes were not allowed to sit in the water bath or at room temperature for any period of time). Cells were diluted into serum-free RPMI containing 30 ⁇ g/ml DNase to prevent clumping by dead cell DNA and washed twice.
  • PBMCs peripheral blood mononuclear cells
  • PBMCs peripheral blood mononuclear cells
  • SAC-I cells expressing Protein A; Calbiochem
  • 20 ⁇ g/ml Immunobeads Rabbit anti-Human IgM; Biorad
  • 20 ng/ml of human rIL-4 Two ml of cells per well were plated in a 24-well plate (Falcon, Becton Dickinson) and cultured at 37°C. After 3 days, the medium was removed and the cells were washed three times followed by addition of RPMI/10% HS. The cells were used after culturing for an additional 2 days in RPMI/10% HS.
  • the SAC-I activated PBMCs were washed once in cold 0.9% sodium chloride (J.T. Baker) containing 1% BSA.
  • the cells were resuspended at 10 7 /ml in cold citrate-phosphate buffer (0.13M citric acid [J.T. Baker], 0.06M sodium phosphate monobasic [Sigma], pH3) containing 1% BSA and 3 ⁇ g/ml ⁇ 2 m and incubated on ice.
  • the cells were irradiated at 6100 rads (5 x 10 6 / ml; 25 million cells/tube), washed, then adjusted to the appropriate concentration for addition to the induction culture (see below).
  • AIS MicroCellector T-150 flasks (specific for the depletion of CD4+ T cells; Menlo Park, CA) were primed by adding 25 ml of PBS/1 mM EDTA, swirling for 30 seconds so that all surfaces were moistened, and then incubating with the binding surface down at room temperature for 1 hour. Following this incubation, flasks were shaken vigorously for 30 seconds, washed 1 time with PBS/EDTA, 2 additional times with PBS and then incubated with 25 ml of culture medium for 15 minutes.
  • PBMCs were thawed in serum-free RPMI (+ L-glutamine + Hepes) containing 30 ⁇ g/ml DNAse, washed once, and incubated for 15 minutes in culture medium. Following aspiration of culture medium from the flasks, up to 180 million PBMCs were added in 25 ml of culture medium containing 30 ⁇ g/ml DNAse. After 1 hour at room temperature, the flasks were rocked gently for 10 seconds to resuspend the nonadherent cells. The nonadherent cell suspension containing the CD8+ T cells was collected and the flasks were washed 2 times with PBS.
  • the CD4+ T cell depleted PBMCs were centrifuged and counted for addition to the induction culture.
  • the CD4+ and CD8+ phenotype of the CD4+ depleted cell population was determined by FACS analysis (see below). In general, this technique resulted in a two-fold enrichment for CD8+ T cells with an average of approximately 40-50% CD8+ T cells and 15-20% remaining CD4+ T cells following depletion of CD4+ T cells.
  • Depletion of CD4+ T cells can also be accomplished by using antibody and complement methods or antibody coated magnetic beads (Dynabeads). Depletion of CD4+ T cells enriched the CTLp and removed cells which competed for cell nutrients.
  • CD4+ depleted PBMC to be used as the responder population were prepared utilizing AIS flasks for selection of CD8+ T cells through the depletion of CD4+ T cells (above).
  • the responder cells were plated at 3 x 10 6 /ml in a 1 ml volume (24 well plate) and placed at 37°C until the peptide loaded stimulator APCs were prepared.
  • the irradiated, peptide loaded APCs were washed 1 time in serum-free RPMI (+ L-glutamine and Hepes), adjusted to the appropriate concentration in complete medium, and plated into a 24 well plate at 1 ml/plate:
  • For PBMC and SAC-I activated PBMCs as APCs 1 x 10 6 stimulator cells (1 ml volume) were plated into the wells containing the responder cells;
  • For PHA blasts as APCs 1 ml of 3 x 10 5 /ml stimulator cells were plated in each well.
  • a final concentration of 10 ng/ml of rIL-7 (2 ml total volume) was added.
  • the PBMCs were resuspended in culture medium and adjusted to 4 x 10 6 /ml and 1 ml of irradiated PBMCs was added per well of a 24-well plate.
  • the PBMC were incubated for 2 hours at 37°C, washed 3 times to remove nonadherent cells, and cultured in medium containing 20 ⁇ g/ml total peptide and 3 ⁇ g/ml ⁇ 2 microglobulin added in a 0.5 ml volume and again incubated for 2 hours at 37°C.
  • the peptide was aspirated and 1.5 x 10 6 responder cells resuspended in culture medium were added in a 1 ml volume.

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Claims (16)

  1. Méthode pour activer des cellules T cytotoxiques in vitro comprenant :
    la dissociation des peptides liés de molécules de MHC classe T sur des cellules présentant un antigène, en utilisant un traitement acide modéré ;
    l'association des peptides immunogènes souhaités avec les molécules de MHC classe I sur les cellules présentant l'antigène ; et
    l'incubation des cellules présentant l'antigène avec les cellules T cytotoxiques en présence d'un facteur de croissance, pour ainsi produire des cellules T cytotoxiques activées.
  2. Méthode de la revendication 1, où l'étape de dissocier les peptides liés est effectuée par incubation des cellules présentant l'antigène dans une solution d'un tampon de glycine ou de citrate-phosphate à pH 3.
  3. Méthode de la revendication 1 ou de la revendication 2, où l'étape d'associer des peptides immunogènes souhaités avec les molécules de MHC est effectuée par incubation des cellules présentant l'antigène avec environ 10 à 50 µg/ml du peptide immunogène.
  4. Méthode de toute revendication précédente où l'étape de soumettre les cellules présentant l'antigène à incubation avec les cellules T cytotoxiques se produit sur une période d'environ 7 à environ 10 jours.
  5. Méthode de toute revendication précédente, où les cellules présentant l'antigène sont des cellules mononucléaires de sang périphérique isolées d'un patient.
  6. Méthode de la revendication 5 où les cellules mononucléaires de sang périphérique sont activées par SAC-I.
  7. Méthode de toute revendication précédente, où le facteur de croissance est IL-7 et ledit facteur de croissance est ajouté au début de l'étape d'incubation et à 7 jours après début de l'étape d'incubation.
  8. Méthode de toute revendication précédente, où le facteur de croissance est IL-2 et ledit facteur de croissance est ajouté 7 jours après le début de l'étape d'incubation.
  9. Méthode de production d'une composition pharmaceutique caractérisée en ce que les cellules T cytotoxiques activées pouvant être obtenues par une méthode des revendications 1-8 sont mises en contact avec un support acceptable.
  10. Méthode de la revendication 9, où les cellules T cytotoxiques activées sont séparées des cellules présentant l'antigène avant contact des cellules T cytotoxiques activées avec un support approprié.
  11. Méthode de la revendication 9 ou de la revendication 10, où la composition pharmaceutique est pour le traitement du cancer, du sida, de l'hépatite, d'une infection bactérienne, d'une infection fongique, de la malaria ou de la tuberculose.
  12. Méthode de production d'une composition pharmaceutique pour tuer spécifiquement des cellules cibles chez un patient humain, comprenant :
    après avoir obtenu un échantillon fluide contenant des cellules T cytotoxiques du patient ;
    la mise en contact des cellules T cytotoxiques avec des cellules présentant un antigène où les cellules présentant l'antigène sont produites par les étapes de :
    a) dissocier les peptides liés des molécules de MHC classe I sur les cellules présentant l'antigène en utilisant un traitement acide modéré ; et
    b) associer des peptides immunogènes souhaités aux molécules de MHC classe I sur les cellules présentant l'antigène ; pour ainsi produire des cellules T cytotoxiques activées ;
    c) la mise des cellules T cytotoxiques activées en contact avec un support acceptable pour ainsi former une composition pharmaceutique.
  13. Méthode de la revendication 12 comprenant de plus l'étape de dissocier les peptides liés des cellules présentant l'antigène par incubation des cellules présentant l'antigène dans une solution d'un tampon de glycine ou de citrate-phosphate à pH 3.
  14. Méthode de la revendication 12 ou 13, comprenant de plus l'étape d'associer des peptides immunogènes souhaités aux molécules de MHC sur les cellules présentant l'antigène par incubation des cellules présentant l'antigène avec environ 10 à 50 µg/ml du peptide immunogène.
  15. Méthode de l'une quelconque des revendications 12 à 14 où les cellules présentant l'antigène sont des cellules mononucléaires de sang périphérique isolées d'un patient.
  16. Méthode de l'une quelconque des revendications 12 à 15 où l'étape d'incubation des cellules présentant l'antigène avec les cellules T cytotoxiques se produit sur une période d'environ 7 à environ 10 jours.
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Families Citing this family (66)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6607727B1 (en) 1991-08-26 2003-08-19 The Scripps Research Institute Peptides for inducing cytotoxic T lymphocyte responses to hepatitus B virus
US5662907A (en) * 1992-08-07 1997-09-02 Cytel Corporation Induction of anti-tumor cytotoxic T lymphocytes in humans using synthetic peptide epitopes
US9340577B2 (en) 1992-08-07 2016-05-17 Epimmune Inc. HLA binding motifs and peptides and their uses
US6235288B1 (en) 1992-08-26 2001-05-22 The Scripps Research Institute Peptides for inducing cytotoxic T lymphocyte responses to hepatitis B virus
WO1994019011A1 (fr) * 1993-02-26 1994-09-01 The Scripps Research Institute Peptides utilises pour induire des reponses aux lymphocytes t cytotoxiques contre le virus de l'hepatite b
AU702517B2 (en) * 1993-08-06 1999-02-25 Epimmune, Inc. Cloning and characterization of the complete MAGE-1 gene
WO1995011255A1 (fr) * 1993-10-19 1995-04-27 Ajinomoto Co., Inc. Peptide pouvant induire une reponse immune contre vih et agent contenant ce peptide pour la prevention ou le traitement du sida
US6682731B1 (en) * 1994-03-24 2004-01-27 Ludwig Institute For Cancer Research Isolated peptides derived from mage tumor rejection antigen precursors which complex with HLA-A2 molecules
US6300090B1 (en) * 1994-07-29 2001-10-09 The Rockefeller University Methods of use of viral vectors to deliver antigen to dendritic cells
US5587289A (en) * 1995-03-14 1996-12-24 Ludwig Institute For Cancer Research Isolated nucleic acid molecules which are members of the MAGE-Xp family and uses thereof
AU7008896A (en) * 1995-08-21 1997-03-12 Duke University A method to increase the density of antigen on antigen presenting cells
AU1394497A (en) 1996-01-17 1997-08-11 Imperial College Innovations Limited Immunotherapy using cytotoxic t lymphocytes (ctl)
US6346390B1 (en) * 1996-03-08 2002-02-12 Receptron, Inc. Receptor derived peptides involved in modulation of response to ligand binding
US6130087A (en) * 1996-10-07 2000-10-10 Fordham University Methods for generating cytotoxic T cells in vitro
US20050170503A1 (en) * 1997-02-27 2005-08-04 University Of Pittsburgh In vitro induction of antigen-specific T-cells using dendritic cell-tumor cell or dendritic cell-viral cell derived immunogens
EP1078092B1 (fr) * 1998-05-13 2011-08-03 Epimmune Inc. Vecteurs d'expression destines a stimuler une reponse immunitaire et procedes de leur utilisation
US7264965B2 (en) * 1998-06-05 2007-09-04 Alexis Biotech Limited Method for producing or enhancing a T-cell response against a target cell using a complex comprising an HLA class I molecule and an attaching means
US7521197B2 (en) * 1998-06-05 2009-04-21 Alexis Biotech Limited Method for producing cytotoxic T-cells
US7713739B1 (en) * 2000-11-17 2010-05-11 Novartis Vaccines And Diagnostics, Inc. Microparticle-based transfection and activation of dendritic cells
US6897288B1 (en) * 1999-10-19 2005-05-24 Ludwig Institute For Cancer Research Mage-A12 antigenic peptides and uses thereof
US6602510B1 (en) 2000-04-05 2003-08-05 Epimmune Inc. HLA class I A2 tumor associated antigen peptides and vaccine compositions
US20070098776A1 (en) * 1999-12-13 2007-05-03 Fikes John D HLA class I A2 tumor associated antigen peptides and vaccine compositions
US7462354B2 (en) * 1999-12-28 2008-12-09 Pharmexa Inc. Method and system for optimizing minigenes and peptides encoded thereby
US20040248113A1 (en) * 1999-12-28 2004-12-09 Alessandro Sette Method and system for optimizing multi-epitope nucleic acid constructs and peptides encoded thereby
CA2433194C (fr) * 2000-10-10 2012-04-03 The Board Of Regents Of The University Of Oklahoma Mappage comparatif de ligand a partir de cellules positives cmh
CA2438505A1 (fr) * 2001-02-14 2002-09-12 Genzyme Corporation Ligands peptidiques modifies
DE10109224A1 (de) * 2001-02-26 2002-09-05 Bayer Ag Flammwidrige Polycarbonat-Zusammensetzungen mit erhöhter Chemikalienbeständigkeit
FR2821947B1 (fr) * 2001-03-12 2003-05-16 Canon Kk Procede et dispositif de validation de parametres definissant une image
US7842480B2 (en) * 2001-05-18 2010-11-30 Mayo Foundation For Medical Education And Research Chimeric antigen-specific t cell-activating polypeptides
US20060222656A1 (en) * 2005-04-01 2006-10-05 University Of Maryland, Baltimore MAGE-A3/HPV 16 peptide vaccines for head and neck cancer
US20030134341A1 (en) * 2001-09-19 2003-07-17 Medcell Biologics, Llc. Th1 cell adoptive immunotherapy
AU2002341717A1 (en) * 2001-09-17 2003-04-01 Medcell Biologics, Llc. Cell therapy system
US20030134415A1 (en) * 2001-09-19 2003-07-17 Gruenberg Micheal L. Th1 cell adoptive immunotherapy
AU2002361559A1 (en) * 2001-09-24 2003-04-28 University Of Pittburgh Of The Commonwealth System Of Higher Education Anticancer vaccine and diganostic methods and reagents
US20030175272A1 (en) * 2002-03-07 2003-09-18 Medcell Biologics, Inc. Re-activated T-cells for adoptive immunotherapy
WO2004030616A2 (fr) 2002-09-17 2004-04-15 Antigen Express, Inc. Vaccins a base d'epitope antigenique ii-key peptidique hybride
US20040072262A1 (en) 2002-10-11 2004-04-15 Montero-Julian Felix A. Methods and systems for detecting MHC class I binding peptides
WO2004076652A1 (fr) * 2003-02-25 2004-09-10 Tokai University Milieu pour cellules souches destinees a etre utilisees pour regenerer un disque intervertebral et regeneration d'un disque intervertebral utilisant des cellules souches
CA2520768A1 (fr) * 2003-03-28 2005-02-10 Idm Pharma, Inc. Procedes d'identification de variants optimaux d'epitopes peptidiques
US20080274129A1 (en) * 2003-04-18 2008-11-06 Fikes John D Hla-A2 Tumor Associated Antigen Peptides and Compositions
US20070298051A1 (en) * 2003-11-19 2007-12-27 Beth Israel Deaconess Medical Center Adjuvants Of Immune Response
US7592431B2 (en) * 2004-02-26 2009-09-22 Immunovative Therapies, Ltd. Biodegradable T-cell Activation device
DK2573166T3 (da) 2004-02-26 2016-07-04 Immunovative Therapies Ltd Fremgangsmåder til fremstilling af T-celler til celleterapi
PL1749090T3 (pl) 2004-03-01 2017-12-29 Immunovative Therapies, Ltd. Sposób formułowania i kompozycja do terapii komórkowej
EP1781313A4 (fr) 2004-06-17 2009-08-26 Beckman Coulter Inc Epitopes de mycobacterium tuberculosis et leurs procedes d'utilisation
WO2007044033A2 (fr) * 2004-12-07 2007-04-19 University Of Pittsburgh Of The Commonwealth System Of Higher Education Récepteur non restreint par le cmh cloné de l'antigène spécifique de tumeur muc1 pour applications thérapeutiques et diagnostiques
US7838503B2 (en) * 2005-06-15 2010-11-23 Children's Medical Center Corporation Methods for extending the replicative lifespan of cells
ES2603418T3 (es) * 2006-10-04 2017-02-27 Janssen Pharmaceutica Nv Preparación de células presentadoras de antígeno artificial inactivado y su uso en terapias celulares
WO2009139921A2 (fr) * 2008-05-16 2009-11-19 Genelux Corporation Microorganismes pour la prévention et le traitement de néoplasmes accompagnant une thérapie cellulaire
WO2010011994A2 (fr) 2008-07-25 2010-01-28 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Polypeptides et leurs utilisations
JP5792630B2 (ja) 2009-01-28 2015-10-14 エピミューン，インコーポレイティド Ｐａｎ−ｄｒ結合ポリペプチドおよびその使用
EP2391748A4 (fr) * 2009-01-28 2012-08-01 Antigen Express Inc Peptides hybrides li-key modulant la réponse immunitaire à la grippe
WO2010123813A1 (fr) * 2009-04-20 2010-10-28 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Épitopes immunogènes de l'antigène ngep
DK2453914T3 (en) 2009-07-16 2018-10-08 Vaxil Biotherapeutics Ltd ANTIGEN-SPECIFIC MULTIPLE PIT-BASED ANTI-INFECTIOUS VACCINES
US8075895B2 (en) * 2009-09-22 2011-12-13 Janssen Pharmaceutica N.V. Identification of antigenic peptides from multiple myeloma cells
US9233156B2 (en) 2011-05-03 2016-01-12 Immunovative Therapies Ltd. Induction of IL-12 using immunotherapy
KR102202460B1 (ko) 2011-05-03 2021-01-14 이뮤노베이티브 테라피스, 엘티디. 생세포를 포함하는 생물학적 약물을 처리하기 위한 방법
WO2013177187A2 (fr) 2012-05-22 2013-11-28 Massachusetts Institute Of Technology Traitement de tumeur synergique avec du pk il-2 étendu et des agents thérapeutiques
JP2017505447A (ja) 2013-12-19 2017-02-16 オペクサ セラピューティクス，インコーポレイティド Ｔ細胞エピトーププロファイリング方法、ｔ細胞組成物の製造方法及び疾患の治療方法
US10993997B2 (en) 2014-12-19 2021-05-04 The Broad Institute, Inc. Methods for profiling the t cell repertoire
US20200215110A1 (en) * 2016-09-09 2020-07-09 The General Hospital Corporation Ex vivo antigen-presenting cells or activated cd-positive t cells for treatment of infectious diseases
CN110809716B (zh) 2017-02-12 2023-07-07 百欧恩泰美国公司 基于hla的方法和组合物及其用途
MX2021007556A (es) 2018-12-21 2021-09-10 Biontech Us Inc Método y sistemas de predicción de epítopos específicos de hla de clase ii y caracterización de células t cd4+.
US20210038684A1 (en) 2019-06-11 2021-02-11 Alkermes Pharma Ireland Limited Compositions and Methods for Cancer Immunotherapy
JP2022539249A (ja) 2019-07-05 2022-09-07 ケース ウェスタン リザーブ ユニバーシティ 幹細胞培養及び幹細胞治療用プライム化培地及びプライム化方法
US20240150711A1 (en) 2021-03-01 2024-05-09 Dana-Farber Cancer Institute, Inc. Personalized redirection and reprogramming of t cells for precise targeting of tumors

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5081029A (en) * 1985-09-25 1992-01-14 Oncogen Methods of adoptive immunotherapy for treatment of aids
US5314813A (en) * 1992-02-19 1994-05-24 Scripps Research Institute Drosophila cell lines expressing genes encoding MHC class I antigens and B2-microglobulin and capable of assembling empty complexes and methods of making said cell lines
DE69326064T2 (de) * 1992-05-26 2000-05-25 Rijksuniversiteit Te Leiden Le Peptide des menschlichen Proteins P53 zum Gebrauch in menschlichen-zytotoxischen-T-Zell-Antwort-induzierenden Kompositionen sowie menschliche P53-Protein-spezifische T-Lymphocyten
IL109664A0 (en) * 1993-05-18 1994-08-26 Rijksuniversiteit Peptides of human influenza virus for use in human t cell response inducing compositions

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EP0726941A4 (fr) 1999-06-16
DE69430315D1 (de) 2002-05-08
US5846827A (en) 1998-12-08
AU7478394A (en) 1995-02-28
EP0726941A1 (fr) 1996-08-21
WO1995004817A1 (fr) 1995-02-16
CA2168950A1 (fr) 1995-02-16

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